Glutamate, an excitatory neurotransmitter/neuromodulator involved in cell-to-cell communication within the central nervous system, is now believed to play a role in neuroendocrine function. In this study we describe a single, saturable, stereospecific, and temperature-, time-, and pH-dependent binding site for glutamate in the pineal gland of the rat (Kd = 612 +/- 23 nM, Bmax = 3.17 +/- 0.33 pmol/mg protein). After removal of the sympathetic innervation to the pineal gland, [3H]glutamate binding displayed a higher apparent affinity (Kd = 412 +/- 28 nM) (P < 0.05) without a change in binding site number (Bmax = 3.60 +/- 0.24 pmol/mg protein). No difference in [3H]glutamate binding site number was observed in pineal glands obtained from animals sacrificed during the middle of the light and dark periods. These data suggest a possible modulatory role for a glutamate binding site in pineal gland function.